Techniques for analyzing biological macromolecules, such as, for example, nucleic acids and proteins, have become increasingly important in the fields of medicine and genetics. One well accepted technique for analyzing biomolecules is gel electrophoresis. In gel electrophoresis a voltage is applied across at least one linear dimension of a medium, typically a liquid buffer or a polymer gel. A sample tagged with a fluorophore is introduced to the medium, and components of the sample separate under the influence of the applied electric field according to their respective electric mobilities. The fluorescently labeled components migrate down the linear dimension of the medium past a station where they are illuminated by a laser beam. Stimulated fluorescent emission from the illuminated components is captured by a detector as a function of time, producing an electropherogram that encodes the analytical information of interest.
Electrophoresis devices are available in a variety of formats. Traditionally, separations are performed in a medium made of cross-linked polymer matrix formed as a gel sheet, or slab gel, between two glass plates. To enable higher applied voltages, remove heat generated by electrophoretic currents, and provide higher throughput, the medium may be confined to narrow glass capillary tubes. Microgrooves fabricated into a planar, laminated substrate of glass or plastic have also been used as conduits for the medium.
In a high throughput analytical device, the capillaries or microgrooves, referred to herein as sample conduits, are arranged in substantially planar arrays so that many samples may be processed at the same time. The array format is most efficient when a single laser, or a small number of lasers, is used to illuminate the capillaries or microgrooves in the array. Since the medium in each conduit absorbs only a tiny fraction of the laser power, most devices utilize an arrangement in which the optical axis of the laser beam output is substantially coplanar with and normal to the longitudinal axes of the conduits. A single laser beam, or, in some cases opposed dual beams, impinge normal to the wall of the first conduit in a substantially planar array, illuminate the fluorescently labeled sample therein, exit the first conduit, propagate to the second conduit, and so forth. This technique has been generally successful for arrays with a small number of conduits, but becomes increasingly unworkable as the number of conduits in the array is increased. The variety of materials in the beam path (for example, glass, medium, air), each having its own index of refraction, as well as the multiplicity of surfaces, creates an extremely complex optical system. Reflection and refraction of the beam at the multiple surfaces diverts the beam from a direct passage though the conduits, which makes efficient and uniform delivery of the light to each conduit problematic.
The need for relative uniformity of illumination stems from the economical practice of using a single detector (or an array of identical detector elements) for measuring signal from each conduit of the planar array. As such, the signal from each conduit, proportional to the intensity of excitation, is detected with the same level of sensitivity and dynamic range. In this arrangement, nonuniform illumination would dictate undesirable trade-offs. For example, adjusting the intensity of the laser beam to achieve maximal sensitivity in a relatively poorly illuminated conduit could lead to detector saturation by signals of other, better illuminated conduits, thereby limiting the dynamic range of the better illuminated conduits. Therefore, array performance is optimized by ensuring that all conduits receive the same intensity of excitation light.
In each of these systems, the array of conduits is treated as a sequential optical system in which all or most of the light energy passing out of one conduit impinges on the next successive conduit in the array. These systems are extremely sensitive to optical misalignment and must be assembled to extremely high tolerances, so manufacturing yields would be expected to be quite low. In addition, this delicate optical system would be easily misaligned if repeatedly handled and installed in an analytical device.
The treatment of conduits as optical elements also places constraints in their geometry, depending on the optical properties of the materials used. For example, for capillaries in a close packed configuration, the ratio of the inner and outer diameters of the capillaries are restricted to a specific range, depending on the refractive indices of the capillary walls, the enclosed medium, and the surrounding medium. Capillaries with dimensions outside these ranges will fail to effectively transmit the beam from one capillary to the next. Optical alignment is not as significant a problem for microgrooves arrays, which may be precisely laid out equidistant from one another on a substrate. However, embossing and chemical etching procedures used to form the microgrooves in the substrate create beveled walls that are not perpendicular to the plane of the array or to the light source. When sealed with a coversheet and filled with a polymer medium, each microgrooves can form a prism-like optical structure that cumulatively causes the beam to deflect out of plane, leaving a majority of the microgrooves insufficiently illuminated.
Previous proposals for array illumination have made unacceptable compromises in illumination intensity or uniformity, or have demanded prohibitive requirements in optical alignment.